Stabilized protease of bacterial origin and method of stabilizing such protease

ABSTRACT

A PROTEASE OF BACTERIAL ORIGIN IS STABILIZED AGAINST THE LOSS OF PROTEOLYTIC ACTIVITY IN THE PRESENCE OF WATER BY COMBINING THE PROTEASE WITH A LESS THAN STOICHIOMETRIC QUANTITY OF AN ENZYME-ION BINDING AGENT TO PROLONG THE SHELF LIFE OF THE ENZYMIC COMPOSITION, AND ACCOMPANYING SAID COMBINATION WITH AT LEAST TWO MEMBERS OF THE GROUP CONSISITING OF SALT, PROTEIN, ORGANIC SOLVENT AND DETERGENT IN A NON-PRECIPITATING QUANTITY.

" ted States 3,575,864 STABILIZED PROTEASE F BACTERIAL ORIGIN AND METHOD OF STABILIZING SUCH PROTEASE Irving Innerfield, 20 Knickerbocker Road, Tenafiy, NJ. 07670 No Drawing. Filed Apr. 17, 1969, Ser. No. 817,172 Int. Cl. Clld 7/42 US. Cl. 25289 11 Claims ABSTRACT OF THE DISCLOSURE BACKGROUND OF THE INVENTION The invention relates to the stabilization of proteinsplitting enzymes of bacterial origin against loss of proteolytic activity in the presence of water and aims to provide a new composition that will exhibit proteolytic activity over an extended period of time even though it is stored under conditions which subject it to the presence of moisture or wherein it is incorporated in an aqueous medium. My new compositions are ideally suited for combination with detergents for laundry and cosmetic use as stabilized liquid detergent agents since detergents do not inhibit but indeed appear to enhance and further stabilize the enzyme action.

Proteolytic enzymes are organic catalysts which depolymerize protein molecules. Thus, proteolytic enzymes can reduce a protein molecule to a chain size sufficiently small to be readily removed by detergents in water or by water alone. Hence, they are useful in the removal of stains which are caused by blood, grass, gravies, wine and the like. It is also known that proteolytic enzymes are useful in oral hygiene and in the treatment of disorders of the skin and body tissues through topical application. However, the use of proteolytic enzymes for such purpose has been severely limited because they normally lack stabilty in aqueous solutions or suspensions. To meet ordinary commercial requirements, the shelf life of a detergent preparation should be twelve or more months. The result has been that proteolytic enzymes designed for laundry purposes have been marketed in dry form and, even then, in opened packages these enzymes absorbed moisture and they have rapidly lost activity thereafter. Furthermore, the shelf life of desirable cosmetic and pharmaceutical preparations, designed for topical application or oral hygiene, which contain proteolytic enzymes of bacterial origin has been far short of the twelve or more months required therefor. Hence, such past prepara tions could not be conveniently marketed.

It is known that proteolytic enzymes can be precipitated out of aqueous solution by adding to such solution a sufiicient quantity of a member of a class of compounds which are referred to hereinafter as enzyme-ion binding agents. Exemplary thereof are trichloracetic acid, tungstic acid, phosphotungstic acid, tannic acid, sulfosalicylic 3,575,854 Patented Apr. 20, 1971 acid; and certain dyes such as methaline blue, safi'ronin and inuline scarlet. The quantity thereof that will precipitate the proteolytic enzyme out of aqueous solution is the amount which is at least the stoichiometric equivalent of that enzyme which is in solution.

I have discovered that stability against loss of more than about 20% of its original proteolytic activity in aqueous medium at room temperature for a period of about three, or more, months can be conferred upon a proteolytic enzyme of bacterial origin, Without precipitating the enzyme out of solution in such medium, by combining with that enzyme a quantity of an enzyme-ion binding agent which is less than the stoichiometric equivalent of the proteolytic enzyme to be stabilized. The more nearly the quantity of enzyme-ion binding agent approaches the stoichiometric equivalent of the enzyme to be stabilized the more eifective it appears to be. The shelf life of the proteolytic enzyme which is thus stabilized can be extended for a further period of upwards of nine months by including in the aqueous solution thereof, in small quantity, at least two members of the group consisting of a salt, an organic solvent, a protein, a detergent. The aqueous medium in which the stabilized proteolytic enzyme is dissolved, which is of high ionic strength and has a low dielectric constant, is maintained at a pH Within the range of from about 7.0 to about 9.5.

SUMMARY The proteolytic enzymes that I employe in the practice of my invention are of bacterial origin. Exemplary thereof are Bacillus subtilis and B. proteus vulgaris. I have discovered that such an enzyme can be stabilized during storage at room temperature for upwards of twelve months with retention of about of its original proteolytic activity in an aqueous medium by combining with that enzyme a slightly less than stoichiometrically equivalent quantity of an enzyme-ion binding agent, and including with that combination a non-precipitating quantity of at least two members of the group consisting of salt, organic solvent, protein and detergent. The aqueous medium containing the stabilized proteolytic enzyme is maintained at a pH within the range of about 7.0 to about 9.5, preferably 7.5 to 8.5, and is most desirably of high ionic strength and has a low dielectric constant.

The shelf life of the stabilized enzyme may be prolonged to the extent necessary to meet commercial requirements (twelve months or more) by adding thereto two or more of the following: a salt (e.g., sodium chloride, ammonium sulfate, sodium sulfate, magnesium sulfate, sodium phosphate, lithium bromide, sodium tannate); a protein (e.g., globulin, preferably gluten); an organic solvent (e.g., ethanol, methanol, acetone, sugar alcohols, linear alcohols, carbocyclic alcohol and glycol), in a quantity which will not precipitate the enzymic material out of solution. It is known that a salt, or an organic solvent or a protein will serve as a precipitating agent when added, in sufiicient quantity, to an aqueous solution of a proteolytic enzyme. Hence, the quantity thereof that may be added to the aqueous solution of stabilized proteolytic enzyme in the practice of my invention must be less than a precipitating quantity thereof. However, the more nearly the quantity of such added material approaches a. precipitating quantity thereof, the more effective it appears to be. The detergents that may be incorporated with advantage in my new stabilized enzymic preparations are alkyl benzene sulfonate, lauryl sulfonate, alkylal'yl sulfonate, sulfonated fatty acids, sodium salt of lauryl ether sulfonate, sodium sulfonate, triethanolamine sulfonate, sulfated fatty acid ester, ammonium salt of sulfate monoglyceride; and non-ionic surface-active agents such as Tween 40, Tween 60 and Tween 80 and Triton.

It appears to me that the extraordinarily long period through which the proteolytic activity of enzymic material is preserved in the presence of moisture through the practice of my invention is due to the fact that the addition of the enzyme-ion binding agent in slightly less than stoichiometric quantity serves to block negatively charged active sites on the enzyme to be stabilized and that the further addition of at least two members of the group consisting of salt, protein, organic solvent and detergent serves to block positively charged active sites on that enzymic material. The net result is that a suflicient number of the reactive groups in the enzyme are immobilized to prevent the proteolytic material from digesting itself, thus preserving its potential proteolytic activity until an additional protein substrate is encountered by it.

In order that my invention may be fully available to those skilled in the art, the following examples of compositions containing proteolytic enzymes of bacterial origin, stabilized against loss of proteolytic activity in an aqueous medium pursuant to my invention, are given:

EXAMPLE 1 Laundry presoak formulation Bacterial protease (derived from B. subtilis)--1 g. Gluten200 mg.

Trichloracetic acidl mg.

Ammonium sulfate60 g.

Water to make 200 ml.

EXAMPLE 2 Stabilized enzymic detergent Bacterial protease (derived from B. subtilis)l g. Gluten-20() mg.

Trichloracetic acid mg.

Ammonium sulfate60 g.

Detergent (alkylaryl sulfonate)-l00 mg.

Water (with pH 7.5 phosphate butter) to make 200 ml.

EXAMPLE 3 Laundry formulation Bacterial protease (derived from B. subtilis)--1 g. Gluten200 mg.

Trichloracetic acidmg.

Sodium chloride g.

Detergent (alkylaryl sulfonate)-1OO mg.

Water (with pH 7.5 phosphate butter) to make 200 ml.

EXAMPLE 4 Shampoo Bacterial protease (derived from B. subtilis)-1 g. Gluten-200 mg.

Trichloracetic acid20 mg.

Detergent (sodium lauryl sulfate)50 mg. Perfume-4 mg.

Water (with pH 7.5 phosphate buffer) to make 200 ml.

EXAMPLE 5 Hand cream Bacterial protease (derived from B. subtilis)-l g. Gluten-200 mg.

Trichloracetic acidl5 mg.

Water (with pH 7.5 phosphate butter) to make 50 m1. Polyethylene glycol (140()0)--5 g.

4 EXAMPLE 6 Dentrifice Bacterial protease (derived from B. subtilis)l g. Trichloracetic acid15 mg.

Sodium lauryl sulfate50 mg.

Sodium metaphosphate-4OO mg.

Dicalcium phosphate-400 mg.

Gum tragacanth10 mg.

Glycerol and water200 ml.

Flavoring material-10 mg.

EXAMPLE 7 Mouth wash Bacterial protease (derived from B. subtilis)l g. Gluten-200 mg.

Trichloracetic acid20 mg.

Triton -400 mg.

Peppermint oil-5 mg.

Spearmint oil5 mg.

Saccharin5 mg.

Sodium carboxymethyl cellulose-5 g.

Water to make 200 ml.

What I claim is:

1. A stabilized proteolytic composition consisting essentially of the combination, in an aqueous medium, with a proteolytic enzyme of bacterial origin of a quantity of enzyme-ion binding agent selected from the group consisting of trichloracetic acid, tungstic acid, phosphotungstic acid, tannic acid, sulfosalicylic acid and dyes selected from the group consisting of methaline blue, saffronin and inuline scarlet which is slightly less than the stoichiome'e ric equivalent of said enzyme and a non-precipitating quantity of at least two stabilizing members of the group consisting of salt, protein, organic solvent for the enzymes and detergent selected from the group consisting of anionic and nonionic surface active agents.

2. A stabilized proteolytic composition as claimed in claim 1 wherein said aqueous medium is of high ionic strength and has a low dielectric constant.

3. A stabilized proteolytic composition as claimed in claim 1 wherein said aqueous medium has a pH within the range from about 7.0 to about 9.5.

4. A stabilized proteolytic composition as claimed in claim 3 wherein said aqueous medium has a pH within the range from about 7.5 to about 8.5.

5. A stabilized proteolytic composition as claimed in claim 1 wherein said ionic binding agent is trichloracetic acid.

6. A stabilized proteolytic composition as claimed in claim 1 wherein said salt is ammonium sulfate.

7. A stabilized proteolytic composition as claimed in claim 1 wherein said protein is gluten.

8. A stabilized proteolytic composition as claimed in claim 1 wherein said detergent is anionic.

9. A stabilized proteolytic composition as claimed in claim 1 wherein said detergent is a non-ionic surfaceactive agent.

10. The method of stabilizing a proteolytic enzyme of bacterial origin in an aqueous medium which comprises combining with said enzyme a quantity of an enzyme-ion binding agent selected from the group consisting of tri chloracetic acid, tungstic acid, phosphotungstic acid, tannic acid, sulfosalicylic acid and dyes selected from the group consisting of methaline blue, safironin and inuline scarlet which is slightly less than the stoichiometric equivalent of said enzyme and adding thereto a non-precipitat ing quantity of at least two stabilizing members of the group consisting of salt, protein, organic solvent for the enzymes and detergent selected from the group consisting of anionic and nonionic surface active agents.

5 6 11. The method of stabilizing a proteolytic enzyme of OTHER REFERENCES bacterial origin as claimed in claim 10 Which includes the 2 step of bufiering said aqueous medium at a pH within the Nature July 2 1967 pages 417 range between about 7.0 to about 9.5. LEON ROSDOL, Primary Examiner References Cited 5 W. E. SCHULZ, Assistant Examiner UNITED STATES PATENTS Us CL XR.

2,343,136 2/1944 Dobson et a1 25289UX 13440; 195-63 3,519,379 7/1970 Blomeyer et a1. 252-89UX 

